Human monoclonal antibody against hepatitis C virus E2 glycoprotein

ABSTRACT

Disclosed is a hybridoma cell line which produces human antibodies capable of binding to the hepatitis C virus (HCV) E2 glycoprotein and capable of neutralizing HCV infection in vivo in an animal model, as well as antibodies produced by the cell line. Also disclosed are various uses of said antibodies in the prevention and treatment of HCV infection. Peripheral blood lymphocytes obtained from human donors having a high titer of anti HCV E2 antibodies are transformed in vitro by Epstein-Barr virus and then fused with heteromyeloma cells to generate hybridomas secreting human antibodies having a high affinity and specificity to HCV E2 glycoprotein.

FIELD OF THE INVENTION

The present invention concerns a hybridoma cell line producing humanantibodies capable of binding to hepatitis C virus envelopeglycoprotein, antibodies produced by the cell line and various usesthereof.

BACKGROUND OF THE INVENTION

Hepatitis C virus (HCV) infection is a major worldwide health problem.Approximately 170 million individuals worldwide are infected by HCV andchronically infected patients carry a high risk of developing cirrhosisand hepatocellular carcinoma (Cohen 1999 Science 285:26–30).

Interferon-α either alone or in combination with ribavirin is used fortherapy of HCV showing efficacy in between 20% and 40% of patientsrespectively.

HCV is an enveloped virus the genetic information for which is encodedin a 9.5 kilo bases positive strand RNA genome. A highly conservednoncoding region of 341 base pairs is localized at the 5′-end of thisviral genome, which is followed by a long open-reading frame coding fora polyprotein of approximately 3,010 amino acids. Two putative envelopeglycoproteins, E1 (gp35) and E2 (gp72) have been identified with 5 or 6and 11 N-linked glycosylation sites, respectively. A high level ofgenetic variability is associated with the envelope genes. This ishighly accentuates at the 5′-end of the E2 gene, where two hypervariableregions termed HVR1 and HVR2, have been described (Kato et al., 1992Bioch. Biophys. Res. Commun. 189:119–127).

Studies using HCV E1–E2 proteins expressed in mammalian cells showedthat infected individuals have an antibody response to HCV E2 (Harada,et al., 1994 J. Gen. Virol. 76:1223–1231). Recent work proposes theexistence of neutralizing antibodies in serum from HCV infected patients(Rosa et al., 1996 PNAS (USA) 93:1759–1763; Zibert et al., 1995 Virology208:653–661; Zibert et al., 1997 J. Virol. 71: 4123–4127).

Investigators employed surrogate assays to provide insights into virusneutralization since the virus cannot be grown in vitro (Houghton.Hepatitis C viruses. In Fields B N, Knipe D M, Howley P M (eds)Virology. Lippincott-Raven, Philadelphia, pp1035–1053). One surrogateassay, the neutralization of binding (NOB) assay, evaluates the abilityof a given antibody or serum to prevent the association of HCV E2protein with a human T-cell line (Rosa et al., 1996 PNAS (USA)93:1759–1763).

Habersetzer et al., 1998 Virology 249:3241 describes human monoclonalantibodies capable of inhibiting the interaction of HCV E2 with humancells in vitro. Burioni et al., 1998 Hepatology 28:810–814 report humanrecombinant Fabs for the HCV E2 protein similarly capable of inhibitingthe interaction of HCV with human cells in vitro.

PCT patent application WO 200005266 discloses antibodies comprising atleast one complementarity determining region (CDR) of the variabledomain of a human antibody that specifically recognize aconformation-dependent epitope of HCV E2 and are capable ofprecipitating E1/E2 complexes.

PCT patent application WO 9740176 discloses a recombinant human antibodyFab portion capable of binding to HCV E2 obtained using a combinatorialantibody library. The relevance of such antibodies for therapy of HCVinfection still needs to be demonstrated.

It is therefore of substantial interest to identify human monoclonalantibodies (Mabs) directed against the E2 glycoprotein that are capableof neutralizing HCV infection in vivo. Such antibodies may constitute anew alternative for the treatment of HCV infections.

Cardoso et al., J. Med. Virol. 55, 28–34 (1998) describes the isolationof human monoclonal antibodies capable of binding to hepatitis C virusenvelope glycoproteins. One of the described antibodies (4F7) wasfurther characterized and sequenced and is the subject matter of thepresent invention.

SUMMARY OF THE INVENTION

In accordance with the present invention, a hybridoma cell line isprovided which secretes human antibodies capable of binding to thehepatitis C virus envelope glycoprotein E2 and capable of neutralizingHCV infection in vivo in an animal model. In accordance with theinvention, peripheral blood mononuclear cells (PBMC) were obtained fromhuman individuals having anti HCV E1/E2 antibodies. PBMC from the humandonor may be obtained either by whole blood donation or byleukophoresis. The human PBMC are then transformed in vitro byEpstein-Barr virus (EBV) (Simoneit at al. 1994 Hybridoma 13:9–13). Aftertransformation the resulting anti HCV producing lymphoblastoid cells arefused in vitro preferably with a human-mouse fusion partner such as aheteromyeloma by techniques well known in the art (e.g. Kohler &Milstein, Nature, 256:495–497, 1975). The generated hybridoma cell linesare either cultured in vitro in a suitable medium wherein the desiredmonoclonal antibody is recovered from the supernatant or, alternativelythe hybridoma cell lines may be injected intraperitoneally into mice andthe antibodies harvested from the malignant ascitis or serum of thesemice. The supernatants of the hybridoma cell lines are screened by anyof the methods known in the art such as enzyme linked immunosorbentassay (ELISA) or radioimmunoassy (RIA) for presence of anti HCV E1/E2antibodies using HCV E1/E2 as a substrate for antibody binding. Thehuman monoclonal anti HCV E1/E2 antibodies thus produced are furtherexamined in a small animal model of HCV infection for their ability toneutralize the virus or reduce the viral load. Virus neutralization orthe reduction in viral load may be measured, for example, by RT-PCRanalysis of HCV RNA in the animal's sera or by the number of HCVpositive mice.

In accordance with the preferred embodiment of the present invention, ahybridoma cell line which was deposited on May 17, 2000, at the EuropeanCollection of Cell Cultures (ECACC, CAMR, Salisbury, Wiltshire, SP4 0JG,UK) under the Accession No. 00051714 is provided. Anti HCV B2 humanmonoclonal antibodies secreted by the above hybridoma cell linedesignated herein as “HCV-AB 68” as well as fragments thereof retainingthe antigen binding characteristics of the antibodies are also provided.Such fragments may be, for example, Fab or F(ab)₂ fragments obtained bydigestion of the whole antibody with various enzymes as known anddescribed extensively in the art. The antigen binding characteristics ofan antibody are determined by using standard assays such as RIA, ELISAor FACS (Fluorescence activated cell sorter) analysis.

The antibodies of the invention have a relatively high affinity to HCVE2 being in the range of about 10⁻⁹ M and 10⁻¹¹ M as determined by aBIAcore 2000 instrument (Pharmacia Biosensor).

The antigen bound by the antibodies defined above also constitutes anaspect of the invention.

Further aspects of the present invention are various prophylactic andtherapeutic uses of the HCV-AB 68 monoclonal antibodies. In accordancewith this aspect of the invention, pharmaceutical compositionscomprising the HCV-AB 68 antibodies may be used for the treatment ofchronic hepatitis C patients by administering to such patients atherapeutically effective amount of the antibodies or fragments thereofcapable of binding to HCV E2. A therapeutically effective amount beingan amount effective in alleviating the symptoms of the HCV infection orreducing the number of circulating viral particles in an individual.Such pharmaceutical compositions may also be used, for example, forpassive immunization of newborn babies born to HCV positive mothers, andfor passive immunization of liver transplantation patients to preventpossible recurrent HCV infections in such patients. A further aspect ofthe invention is a pharmaceutical composition comprising atherapeutically effective amount of the antibodies of the inventioncombined with at least one other anti viral agent as an additionalactive ingredient. Such agents may include but are not limited tointerferons, anti HCV monoclonal antibodies, anti HCV polyclonalantibodies, RNA polymerase inhibitors, protease inhibitors, IRESinhibitors, helicase inhibitors, immunomodulators, antisense compoundsand ribozymes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph of a Western Blot showing binding of HCV-AB 68 toseveral E2 proteins treated under various conditions prior toelectrophoresis. Panel (a) and (b) represent E2 samples prepared undernative conditions. Panel (c) and (d) represent E2 samples treated withβ-mercaptoethanol (β-ME). Panel (e) and (f) represent E2 samples treatedwith DTT. Panel (a) (c) (e) represents E2 samples that were incubatedfor 5 minutes in 37° C. prior to loading on the gel while in panel (b)(d) (f) samples were heated in 100° C. prior to loading on the gel.

Individual lanes contain proteins expressed from different E2 constructsor by using different expression systems:

-   1 E2 BEVS. A recombinant E2 protein produced in insect cells.-   2 E2 BEVS without HVR1 (hyper variable region 1). A recombinant E2    protein lacking 33 amino acids at the 5′ end.-   3 E2 MCS BEVS. A recombinant E2 protein representing the most common    sequences from all 6 genotypes of HCV.-   4 E2 MCS BEVS without HVR1. A recombinant E2 protein as in 3 that    lacks most of the HVR1 except for 4 amino acids.-   5 E2 CHO. A recombinant E2 protein produced in CHO cells.

FIG. 2 demonstrates Immuno Magnetic Separation (IMS) of HCV particlesfrom HCV-infected sera by HCV-AB 68 and a control antibody (HCV-AB17).FIG. 2A is a photograph of an agarose gel showing PCR productsrepresenting HCV particles. FIG. 2B is a graphic representation of thepercentage of bound virus out of the total amount of virus detected byPCR in the bound+unbound fractions.

FIG. 3 represents the nucleic acid sequence of the light (SEQ ID NO:2)and heavy (SEQ ID NO:1) chains of the variable domain of HCV-ABG68. TheFWs (Frameworks) and CDRs (complementarity determining regions) of thegenes are marked.

FIG. 4 represents the amino acid sequence of the light (V_(L)) (SEQ IDNO:3) and heavy (V_(H)) (SEQ ID NO:4) chains of the variable domain ofHCV-AB 68. The FWs and CDRs of the genes are marked.

FIG. 5 is a graphic representation of the mean viral load and percentageof HCV infected animals (numbers in parentheses) at day 12 aftertransplantation in the untreated group, HCV-AB 68 treated group and in acontrol group treated with HBV-AB 17 (in the treatment mode).

FIG. 6 is a graphic representation of the mean viral load and percentageof HCV-Trimera mice with positive HCV RT-PCR signal in their serum(numbers in parentheses) at days 16 and 21 after transplantation in theuntreated group and in the groups treated with different doses of HCV-AB68.

FIG. 7 is a graphic representation of the mean viral load and percentageof HCV-Trimera mice with positive HCV RT-PCR signal in their serum(numbers in parentheses) at day 11 after transplantation in theuntreated group, HCV-AB 68 treated group and in a control group treatedwith HBV-AB 17 (in the inhibition of infection mode).

FIG. 8A is a photograph of a Western Blot showing binding of anti HCVantibody 4E5 to several E2 proteins expressed from different E2constructs or by using different expression systems. Lane numberscorrespond to those described in FIG. 1.

FIG. 8B is a graphic representation of the mean viral load andpercentage of HCV-Trimera mice with positive HCV RT-PCR signal in theirserum (numbers in parentheses) at day 15 after transplantation in theuntreated control group, HCV-AB 68 treated group and in a group treatedwith 4E5 (in the inhibition of infection mode).

FIG. 9 is a graphic representation of the mean viral load and percentageof HCV-Trimera mice with positive HCV RT-PCR signal in their serum(numbers in parentheses) at day 14 after transplantation in theuntreated group, the group treated with I70, the group treated withHCV-AB 68 and the group treated with both I70 and HCV-AB 68.

Reference will now be made to the following Examples that are providedby way of illustration and are not intended to be limiting to thepresent invention.

EXAMPLES

Materials and Methods

EBV Transformation and Cell Fusion

Peripheral blood mononuclear cells (PBMC) were obtained from an HCVinfected human blood donor. The donor was shown to be positive for HCVin a third-generation ELISA (Ortho Diagnostic Systems, Germany) and alsoin a supplemental test (RIBA, Ortho or Matrix, Abbott). PBMC from thisdonor were transformed with Epstein-Barr virus (EBV) (Siemonet K et al.1994, Hybridoma 13: 9–13). The supernatants of the Lymphoblastoid cellsproduced by the transformation were screened for anti HCV E1/E2 antibodyproduction. The screening was performed by an ELISA using the envelopeproteins E1 and E2 of an HCV genotype 1a virus (H strain). Positiveclones were fused with the heteromyeloma cell line K6H6/B5 (Cardoso M Set al. 1998, J Med Virol 55: 28–34). The resulting hybridoma clones werescreened as above and the positive ones were cloned three times bylimiting dilution and finally expanded for antibody production.

Determination of IgG Subclass:

Human IgG subclass was determined by sandwich ELISA using plates coatedwith either purified goat F(ab)₂ anti-human IgG (Chemicon) (0.25μg/well) or E2 (produced in-house by a Baculovirus expression system)(0.1 μg/well). Mouse anti-human IgG subclasses (IgG1, IgG2, IgG3, IgG4)(Sigma) were used as second antibodies and purified goat anti-mouse IgGperoxidase-conjugate (Zymed Laboratories) as the detection reagent. Onehour after incubation at room temperature and washings, substratesolution 3,3′,5,5′-tetramethyl-benzidine dihydrochloride (TMB) (Sigma)was added. Results were read using an ELISA reader at 450 nm.

Affinity Constant Measurements

The affinity constant (KD) of purified HCV-AB 68 to HCV E2 wasdetermined by the BIAcore 2000 instrument (Pharmacia Biosensor)according to the manufacturer's instructions, using BIAevaluation 3.0software provided by the manufacturer to determine the dissociationrates.

HCV E2 proteins from two different sources were used: 1) E2 CHO, acommercially available recombinant HCV genotype 1b E2 protein producedin mammalian CHO cells (Austral Biologicals, CA; based on Choo et al.1991 PNAS 88: 2451). It is a truncated protein, comprising of 331 aminoacids and electrophoreses as a band of ˜60 kD on Western blots. And 2)E2 BEVS, recombinant HCV genotype 1b E2 protein produced in insect cellsusing Baculovirus as an expression vector. The protein comprises 279amino acids and lacks 150 amino acids at the 3′ hydrophobic end of E2(These amino acids were deleted in order to render the protein solubleand enable its expression)

Western Blot Analysis

All protein gels were performed using ready-made 4–12% NuPage Bis-Trisgels (Novex, CA). Protein transfer to nitrocellulose was performed usinga Xcell II mini-cell (Novex, CA) according the manufacturer'srecommendations.

For native gels, 200 ng of each antigen was mixed with a native samplebuffer devoid of reducing agent and incubated for 5 min at 37° C. or100° C. prior to loading. For reducing gels, identical amounts ofantigen were mixed with a native sample buffer containing eitherβ-mercaptoethanol (360 mM) or DTT (50 mM) and processed in an identicalmanner as for native gels.

Following transfer, blots were incubated overnight in blocking buffer(PBS/Milk/0.04% Tween-20) and thereafter incubated with 0.5 μg/ml HCV-AB68 for 3 hrs. The blots were washed three times with blocking buffer andthen incubated with peroxidase conjugated goat-anti human IgG diluted1:20,000 in blocking buffer for 60 min. Following 3×5 min washes withPBS/0.04% Tween-20, blots were exposed to X-ray film and developed usingenhanced chemiluminescence (ECL).

ELISA:

Microtiter plates (F96 Maxisorp Nunc-Immuno Plate, Nunc, Denmark) werecoated with E2-BEVS at 2 μg/ml in PBS, 50 μl/well. Purified HCV-AB 68serially diluted from 1 μg/ml was added to the wells and incubated O.N.at 4° C. Goat anti-human IgG peroxidase-conjugate (Zymed) diluted1:20,000 was used as 2^(nd) antibody. One hour after incubation at 37°C. and washings, substrate solution 3,3′, 5,5′-tetramethyl-benzidinedihydrochloride (TMB, Sigma, St. Louis, Mo) was added. Results were readusing an ELISA reader, with a wavelength of 450 nm.

Immunoprecipitation of HCV from Human Infected Sera

To determine the ability of HCV-AB 68 to bind HCV particles, an immunomagnetic separation (IMS) assay has been developed. Viral HCV particlespresent in sera taken from infected patients are captured by magneticbeads coated with a specific antibody. Following magnetic separation ofbound to non-bound fractions, these particles are detected by a specificRT-PCR assay.

2 μg of HCV-AB68 were coated on protein-A magnetic beads (Dynal A.S.)according to manufacturer instructions. HBV-AB 17, a monoclonal antibodyraised against HBV served as a negative control. All reactions wereperformed at room temperature. Antibody coated beads were washed 3 timesin 0.1 M Na—P buffer pH=8.1, blocked for 30 minutes in 1% BSA, andwashed again in PBS before re-suspension in PBS-0.1% BSA. In parallel,tested serum from an infected individual was pre-treated with protein-Asepharose (Pharmacia) to eliminate serum antibodies. This was achievedby incubating 10 μl serum with 10 μl protein-A sepharose for 30 minuteswith shaking, followed by a 5 minutes 5000 RPM centrifugation step. Theantibody depleted serum was then incubated with shaking for 2 hours withthe antibody coated magnetic beads. PBS containing 0.1% BSA was used tocomplete the final volume to 200 μl. The bound fraction, magneticallyseparated from the non-bound fraction, was washed 5 times with 1 ml PBSbefore final re-suspension in 200 μl PBS.

Evaluation of viral amounts in the bound and non-bound fractions wasperformed by RT-PCR analysis. Viral RNA was extracted using Tri-ReagentBD (Sigma) according to manufacturer instructions. RT reaction (20 μlfinal volume) contained 4 μl RT buffer, 1mM dNTP's, 10 mM DTT, 100 UmMLV-RT (Promega), 2.7 U ANV-RT (Promega) and 2.5 pM HCV anti-senseprimer ATGRTGCTCGGTCTA (SEQ ID NO:5). Reaction conditions were set toramping from 37° C. to 42° C., with a 1° C. increment every 20 minutes.Reaction was completed by a 10 minutes incubation step at 94° C. 5 μl ofRT reaction was used as a template for a PCR reaction (50 μl finalvolume). PCR reactions contained 5 μl PCR buffer, 2.5 mM MgCl₂, 0.2 mMdNTP's, 0.25 U Taq polymerase (Promega), 0.25 pM sense primerCACTCCACCATRGATCACTCCC (SEQ ID NO:6) and anti-sense primerACTCGCAAGCACCCTATCAGG (SEQ ID NO:7). Thirty three amplification cyclesof 1 minute at 94° C., 1 minute at 58° C., 1.5 minute at 72° C. wereperformed, with a final 5 minute elongation step at 72° C. PCR productswere separated on a 2% agarose gel, visualized and quantified followingEtBr staining on an EagleEye II device.

Sequence Analysis RNA was isolated from 10×10⁶ hybridoma cells usingRNAsol B (TEL-TEX, Inc.). cDNA was prepared from 10 μg of total RNA withreverse transcriptase (RT) and oligo dT (Promega) according to standardprocedures in 50 μl reaction volume. PCR was performed on 1 μl of the RTreaction mixture with the following degenerate primers:

5′ leader primers: Heavy chain (V_(H)): (SEQ ID NO:8)5′ GGGAATTCATGGAGTTKGGGCTKAGCTGGRTTTTC 3′ Light chain (V_(λ):) (SEQ IDNO:9) 5′-GGGAATTCATGRCCTGSWCYCCTCTCYTYCTSWYC-3′ Or (V_(κ):) (SEQ IDNO:10) 5′-GGGAATTCATGGACATGRRRDYCCHVGYKCASCTT-3′ (SEQ ID NO:11) The3′ primers correspond to human constant regions: V_(H) 5′-GCGAAGCTTTCATTTACCCRGAGACAGGGAGAG-3′ (SEQ ID NO:12) V₈₀ 5′-GCGAAGCTTCTATGAACATTCTGTAGGGGCCAC-3′ (SEQ ID NO:13)V_(κ) 5′-GCGAAGCTTCTAACACTCTCCCCTGTTGAAGCTC-3′.

Commonly used single letter codes were incorporated into the primersequence:

-   R=A or G S=C or G W=A or T Y=C or T D=A or G or T H=A or C or T V=A    or C or G K=G or T

The full-length heavy and light chains' cDNA (the PCR reaction products)were cloned into a mammalian expression plasmid. Multiple independentcombinations of heavy and light chain DNAs were transfected into tissueculture cells and the supernatant was taken for antibody specificactivity assay. Positive combination of heavy and light chain plasmidsi.e. a combination that resulted in antibody activity was sequenced.Sequences were analyzed by comparison to Genbank and by alignment toKabat sequences (Kabat et al., 1991, Sequences of proteins ofimmunological interest (5^(th) Ed.) U.S. Dept. of Health Services,National Institutes of Health, Bethesda, Md.).

Example 1

Peripheral blood mononuclear cells (PBMC) from human donors positive foranti HCV antibodies were obtained and transformed in vitro with EBV asdescribed above. The cells were than fused with a human mouseheteromyeloma to form hybridoma cell lines. One of the stable hybridomaclones secreting specific human anti HCV E1/E2 antibodies designatedHCV-AB 68 was further characterized. The antibodies secreted by theabove clone were found to be IgG1. The affinity constant of HCV-AB 68 toE2 CHO was shown to be 1.4×10⁻¹¹±0.3, and to E2 BEVS 1.7×10⁻⁹±1.Specificity was tested by Western Blot analysis and by ELISA. In WesternBlot HCV-AB 68 binds different E2 constructs expressed both in abaculovirus expression system (BEVS) and in a mammalian expressionsystem (CHO cells). FIG. 1 demonstrates that HCV-AB 68 binds to all E2constructs except for construct no. 2, i.e. E2 protein without the HVR1region. The antibody does not bind to E2 in the presence of βmercaptoethanol or DTT. These results indicate that HCV-AB 68 recognizesa conformational epitope.

Furthermore, the antibody does not bind to construct 2 (which lacksHVR1) but does bind to construct 4 (lacking HVR1 except for 4 aminoacids at the 3′ end of HVR1, IQLI). It seems that these amino acids forma part of the antibody's binding epitope on E2. Table 1 demonstrates thebinding characteristics of HCV-AB 68 to E2 BEVS in ELISA.

TABLE 1 Binding of HCV-AB 68 to E2 by ELISA HCV-AB 68 μg/ml OD_(450 nm)1 0.439 0.5 0.362 0.25 0.305 0.125 0.267 0.0625 0.204 0.03125 0.1710.0156 0.12 0.0078 0.065

Example 2

The HCV-AB 68 antibodies were used to capture HCV particles from theblood of HCV infected patients by immuno magnetic separation (IMS) asdescribed in materials and methods. Two patients were tested inindividual experiments where each experiment was performed intriplicate. The HCV genotype of patient 1 is 1b while the genotype ofpatient 2 is 1a/1b. Each serum has an HCV titer of 1–5×10⁶. FIG. 2 showsthat the fraction bound by HCV-AB68 is significantly larger then thefraction bound by the control HBV-AB17. HCV-AB68 can bind more than 50%of the viral particles, while the control HBV-AB17 background binding isbetween 1–5% (FIG. 2B).

Example 3

The genes encoding the variable regions of HCV-AB 68 were isolated,fully sequenced and subgroups and complementarity determining regions(CDRs) were determined. The antibody has a fully human Ig gene sequenceas determined by alignment to Genebank sequences and Kabat proteinsequences. FIG. 3 shows the nucleotide sequence of the cDNA encoding theheavy chain and the light chain of the variable region of HCV-AB 68(Sequence identification numbers 1 and 2 respectively). FIG. 4 shows thecorresponding amino acid sequence (Sequence identification numbers 4 and3 respectively).

The sequencing data revealed that the variable region of HCV-AB 68consists of the subgroups V_(H3) J_(H4) V_(K1) J_(K1).

Example 4

The biological activity of HCV-AB 68 was characterized using thefollowing HCV-Trimera animal model: a mouse was treated so as to allowthe stable engraftment of human liver fragments. The treatment includedintensive irradiation followed by transplantation of scid (severecombined immuno deficient) mice bone marrow. Viral infection of humanliver fragments was performed ex vivo using HCV positive human serum(U.S. Pat. No. 5,849,987).

The animal model was used in two different modes representing variouspotential uses of the antibodies: treatment and inhibition of infection.

-   1. Treatment—This mode demonstrates the ability to use the antibody    to treat chronic HCV infection. Trimera mice with established HCV    viremia were administered two intraperitoneal injections of HCV-AB    68 or the non-relevant HBV-AB 17; a total of 40 μg mAb per mouse,    given at two consecutive days (20 μg/mouse/day). HCV-RNA was    determined in mice sera sampled one day after treatment completion.    FIG. 5 demonstrates that HCV-AB 68 was able to reduce both the    percentage of positive mice (from 85% to 42%) and the mean viral    load (from 3.1×10⁴ to 1.0×10⁴ HCV RNA copies/ml). The non-relevant    human monoclonal antibody, HBV-AB 17, did not show any ability to    reduce the mean viral load or the percentage of positive mice. FIG.    6 shows that the effect of HCV-AB 68 in reducing viral load and    percentage of HCV-positive mice is dose dependent. A total dose of 5    μg/mouse reduced the viral load from 4.2×10⁴ to 1.6×10⁴ HCV-RNA    copies/ml mouse serum and the percentage of HCV-RNA positive mice    from 71% to 43% as measured 1 day post treatment. The highest dose    (60 μg/mouse) demonstrated the strongest effect and a reduction in    the viral load by more than 1-log factor (from 4.2×10⁴ to 3.1×10³    copies/ml) and the percentage of HCV-positive mice from 71% to 14%.    Six days after treatment cessation, the viral loads and the    percentages of HCV-positive mice, for all 3 doses, bounced somewhat    back to levels significantly lower than the levels seen in the    untreated control group. At this point in time, a dose response    behavior was still observed for the mean viral loads of the    different groups.-   2. Inhibition of infection—This mode demonstrates the ability to use    the antibody to prevent liver infection. 0.5 ml samples of human    sera containing 10⁶ HCV-RNA copies/ml were pre-incubated with 200 μg    of HCV-AB 68 or with the non-relevant human anti-HBsAg monoclonal    antibody HBV-AB 17 (for 3 h at room temperature) and subsequently    used to infect normal human liver fragments ex vivo. Following    infection, the liver fragments were transplanted in mice and HCV-RNA    was determined in sera 11 or 15 days later. FIG. 7 shows the effect    of HCV-AB 68 in inhibiting liver infection by HCV, as demonstrated    by both the mean viral load and the percentage of HCV-RNA positive    mice. HCV-AB 68 reduced the percentage of these mice from 75% to    41%. It also reduced the mean viral load from 2.2×10⁴ to 5.0×10³    HCV-RNA copies/ml mouse sera. An equal amount (200 μg) of the    non-relevant human monoclonal antibody, HBV-AB17, was not able to    reduce neither the percentage of positive mice nor the viral load    and gave results that are similar to the untreated control group    (FIG. 7). In a different experiment, HCV-AB 68 was able to reduce    the percentage of HCV positive mice from 76% to 41% (p=0.05, n=17)    and the mean viral load from 6.0×10⁴ to 1.1×10⁴ HCV-RNA copies/ml    (p=p.003) as measured in sera that was sampled 15 days post    transplantation.

In contrast, another anti HCV human monoclonal antibody 4E5 capable ofbinding to E2 in vitro as demonstrated by Western blot analysis (FIG.8A), failed to reduce the percentage of HCV infected mice and the meanviral load in the HCV-Trimera animal model (FIG. 8B).

Example 5

The therapeutic effects of a combination of two different potentialantiviral agents were tested in the HCV-Trimera model. The experimentincluded four groups of mice. One group was treated with the antiviraldrug I70 (obtained from Biochem Therapeutics, a small molecule whichinhibits HCV translation). The second group with HCV-AB 68. The thirdgroup with a combination of both. And a fourth group served as a controland received only Dulbecco's PBS. In the 170 treatment group each of themice received 1 mg of the drug at days 10–13 post-liver transplantation(0.25 mg/mouse/day given intraperitoneally (i.p.)). In the HCV-AB 68treatment group each mouse received 40 μg of the antibody at days 12–13post-liver transplantation (20 μg/mouse/day i.p.). In the combinationgroup each mouse received a combination of the above treatments. Viralload in mice sera and percentage of HCV-positive mice were determined atday 14 (one day after treatment). FIG. 9 illustrates the therapeuticadvantage of using a combination of drugs. Treatment with I70 reducedthe HCV mean viral load from 4.72×10⁴ to 1.46×10⁴ and the percentage ofHCV-positive mice from 93% to 50%, whereas that with HCV-AB 68 reducedthe HCV mean viral load to 1.74×10⁴ and the percentage of HCV-positivemice to 50% as measured 1 day post-treatment. The combination treatmentreduced the viral load to 5.83×10³ and the percentage of HCV infectedanimals to 28%.

1. A human monoclonal antibody or fragment, capable of binding to HCVenvelope glycoproteins and capable of neutralizing HCV infection invivo, being selected from the group consisting of: (a) a humanmonoclonal antibody HCV-AB 68, which is secreted by the hybridoma cellline deposited in the European Collection of Cell Cultures (ECACC) underAccession No. 00051714, or a fragment thereof which retains the antigenbinding characteristics of HCV-AB68; and (b) a human monoclonal antibodyor fragment thereof comprising at least a heavy chain variable regionwhose amino acid sequence is depicted in FIG. 4 (SEQ ID NO: 4) and alight chain variable region whose amino acid sequence is depicted inFIG. 4 (SEQ ID NO: 3).
 2. The hybridoma cell line deposited at the ECACCon May 17, 2000 under Accession No.
 00051714. 3. A pharmaceuticalcomposition comprising a therapeutically effective amount of theantibody of claim 1 and a pharmaceutically acceptable carrier.
 4. Apharmaceutical composition comprising a therapeutically effective amountof the antibody of claim 1 combined with at least one other antiviralagent as an additional active ingredient.
 5. A method for the treatmentof HCV infections comprising administering to an individual in needthereof a therapeutically effective amount of antibodies according toclaim
 1. 6. A method for reducing the occurrence of HCV infections in apopulation of individuals, comprising administering a human monoclonalantibody in accordance with claim 1 to a population of individuals toreduce the occurrence of HCV infections in the population.
 7. A methodfor the treatment of HCV infections comprising administering to anindividual in need thereof a therapeutically effective amount of apharmaceutical composition according to claim
 3. 8. A method for thetreatment of HCV infections comprising administering to an individual inneed thereof a therapeutically effective amount of a pharmaceuticalcomposition according to claim
 4. 9. A polypeptide that is the lightchain of the variable region of monoclonal antibody HCV-AB 68 having theamino acid sequence illustrated in FIG. 3, or a portion thereofmaintaining the HCV binding specificity of monoclonal antibody HCV-AB68.
 10. A polypeptide that is the heavy chain of the variable region ofmonoclonal antibody HCV-AB 68 having the amino acid sequence illustratedin FIG. 3, or a portion thereof maintaining the HCV binding specificityof monoclonal antibody HCV-AB
 68. 11. The pharmaceutical composition ofclaim 4, wherein said antiviral agent is selected from the groupconsisting of interferons, anti-HC monoclonal antibodies, anti-HCpolyclonal antibodies, RNA polymerase inhibitors, protease inhibitors,IRES inhibitors, helicase inhibitors, antisense compounds and ribozymes.